Fig 2: NUCKS1 is required for the restart of replication forks after fork stalling and fork collapse. (A) Schematic of the DNA labeling protocol used in (B) and (C). (B) Knockdown of NUCKS1 causes an increase in only red-labeled fibers after HU treatment. U2OS cells were transfected with one of two different siRNAs for NUCKS1 (N #2, N #3), or with negative control (neg. con.), with RAD51AP1 (AP1) or with PALB2 (P #2) siRNA. The data are the means ± 1 SEM of three independent experiments. The bar graphs show the fraction of red-labeled fibers after HU treatment, normalized to the untreated control for each gene-specific knockdown. *P < 0.05; and **P < 0.01; n.s, not significant. (C) Knockdown of NUCKS1 causes a reduction of DNA elongation rates after HU treatment. U2OS cells were transfected with one of two different siRNAs for NUCKS (N #2, N #3), or with negative control (neg. con.), RAD51AP1 (AP1) or PALB2 siRNA (P #2). IdU elongation rates were determined by measuring the green (IdU) of both red- and green-labeled DNA fibers using Image J, and fiber lengths were converted into kb/min, as described (48). IdU elongation rates are presented as the means ± 1 SEM of three independent experiments. ***P < 0.001 (D) Schematic of the DNA labeling protocol used in (E)–(G). (E) Knockdown of NUCKS1 causes an increase in stalled replication forks after topotecan (TPT) treatment. Red-labeled fibers were quantified using Image J. The normalized data (to untreated cells of each gene-specific knockdown condition) are represented as the means ± 1 SEM of at least three independent experiments. *P < 0.05. (F) After TPT treatment, NUCKS1-depleted cells show faster elongating IdU tracts compared to unperturbed NUCKS1-depleted cells. IdU elongation rates were determined by measuring green tracts (IdU) of both red- and green-labeled DNA tracts using ImageJ and tract lengths were converted into kb/min, as described (48). The data are represented as means ± 1 SEM of three independent experiments. The bar graphs show the IdU elongation rates for untreated cell populations and after TPT treatment. ***P < 0.001; n.s., not significant. (G) Knockdown of NUCKS1 causes an increase in second pulse origin firing after TPT treatment. Second pulse origins were determined by quantification of green tracts in both red- and green-labeled DNA tracts using ImageJ. The data shown are the means ± 1 SEM of three independent experiments. **P < 0.01; n.s., not significant.

Fig 3: RAD51AP1 interacts with the nucleosome core particle (NCP).A, schematic of the NCP and the EMSA protocol. B, EMSA of RAD51AP1-His6 (0.1 and 0.2 µM) with the NCP (0.2 µM) assessed by ethidium bromide (lanes 3–5) first and then by Imperial protein stain (lanes 3–5). The western blots show the presence of RAD51AP1 entering the gel only when bound to the NCP (lanes 6–7), not alone (lane 8). C, EMSA of His6/FLAG-tagged RAD51AP1 (0.4 and 0.8 µM) with the NCP (0.4 µM) visualized by ethidium bromide (lanes 1–4). A second gel was loaded in duplicate, transferred, and probed to histone H2A or RAD51AP1. D, EMSA of His6/FLAG-tagged RAD51AP1 (0.4 and 0.8 µM) with the NCP (0.4 µM) visualized by ethidium bromide (lanes 1–3). A second gel was loaded in duplicate, transferred, and probed to histone H3 or RAD51AP1. E, EMSA of His6/FLAG-tagged RAD51AP1 (0.1 and 0.2 µM) and of FLAG-tagged RAD54 (0.1 and 0.2 µM) with the NCP (0.2 µM) assessed by ethidium bromide (lanes 3–4 and 5–6, respectively). F, RAD51 does not bind to the NCP. EMSA of RAD51 and RAD51AP1 (0.4 and 0.2 µM, respectively) with the NCP (0.2 µM).

Fig 4: Wild type levels of both NUCKS1 and RAD51AP1 are required to prevent increased pRPA(S4/S8) levels, indicative of increased replication stress, after a 24 h low dose camptothecin (CPT) treatment. (A) Western blots to show increased levels of pRPA(S4/S8) in NUCKS1-depleted (upper panel) and RAD51AP1-depleted (lower panel) U2OS cells, when compared to U2OS cells transfected with negative control siRNA, immediately (0 h) and at 24 h after a 24-h incubation in 20 nM CPT. (B) Analysis of the cell cycle distributions shows a larger fraction of G2/M-phase cells for NUCKS1- and RAD51AP1-depleted cells than in control cells transfected with negative control siRNA at 24 h post CPT wash out. Bars represent the means from 3 independent experiments ± 1 SEM. *P = 0.05.

Fig 5: NUCKS1 and RAD51AP1 are paralogs. (A) ClustalW multiple sequence alignment of human NUCKS1 and human RAD51AP1. There is a 28% amino acid sequence identity (black) and 11% similarity (gray) between both proteins. The C-terminal domain (encompassing the last 25 amino acids), identified in human RAD51AP1 to facilitate the interaction with RAD51 (52), is underlined. Critical residues within the domain, as identified by mutational analysis (52), are shown in boxes. (B) ClustalW multiple sequence alignment of NUCKS1 and RAD51AP1 from Xenopus tropicalis. These two proteins share 29% identity (black) and 11% similarity (grey).